PROJECT SUMMARY Sepsis continues to be a formidable public health problem with high morbidity and mortality, and there is a need to further define the basic mechanisms of sepsis for future development of novel therapeutics. Our studies will define the anti-inflammatory mechanisms and effects on sepsis outcomes of two endovanilloids, N-arachidonoyl dopamine (NADA) and N-oleoyl dopamine (OLDA), and their receptor, the transient receptor potential vanilloid 1 (TRPV1). NADA activates cannabinoid receptors (CBRs) so is also an endocannabinoid. We found that NADA decreases inflammation and mortality in endotoxemic mice, reduces inflammation in a cecal ligation and puncture model of sepsis (CLP sepsis), and decreases activation of endothelial cells by LPS, bacterial lipopeptides, and TNF?. Using bone marrow chimeras of Trpv1-/- and wild-type mice we determined that NADA reduces inflammation via TRPV1 expressed by non-hematopoietic cells. We also observed that, splenectomy reduces NADA's anti-inflammatory effects in endotoxemic mice, which suggests that NADA is acting via the cholinergic-anti-inflammatory pathway, which downregulates inflammation through the vagus nerve and splenic release of acetylcholine, which reduces endothelial cell and leukocyte inflammation. NADA also affects plasma levels of inflammatory neuropeptides in endotoxemic mice, reducing CGRP and increasing Substance P. Our preliminary data suggest that the CBRs may also contribute to NADA's anti-inflammatory effects. These observations raise several important questions that we will answer in our Aims: (1) What cells regulate the TRPV1-dependent anti-inflammatory effects of NADA and other TRPV1 agonists? (2) Do NADA, OLDA and TRPV1 regulate inflammation via neuro-immune mechanisms? (3) Do NADA, OLDA and TRPV1 affect clinical outcomes of sepsis, and how does co-activation of TRPV1 and CBRs affect sepsis outcomes? Specific Aim #1 will test the hypotheses that neuronal and endothelial cell TRPV1 activation reduces inflammation in endotoxemia and CLP sepsis, and contributes to the anti-inflammatory effects of NADA and OLDA. We will use mice that have undergone ablation of TRPV1 neurons, or knockdown of neuronal and endothelial cell TRPV1, as well as human and mouse endothelial cells activated with TLR agonists. Specific Aim #2 will test the hypothesis that neuro-immune mechanisms regulate NADA's anti-inflammatory effects in sepsis, focusing on the cholinergic anti-inflammatory pathway, and on CGRP and Substance P. Specific Aim #3 will test the hypothesis that NADA and OLDA affect clinical sepsis outcomes, including organ injury, survival and bacterial clearance, and that the activation of CB1R and/or CB2R modulates the TRPV1- dependent anti-inflammatory effects of NADA in sepsis. These studies will advance the understanding of the endovanilloid and endocannabinoid systems in sepsis, with the ultimate goal to identify novel therapeutic targets for sepsis. The results will also have implications for other inflammatory syndromes, such as autoimmune diseases, trauma, ischemia reperfusion injury, and pain.